Humoral immunity provides protection from pathogenic viral, bacterial, and parasitic infections and is mediated by antibodies that are produced following the differentiation of B cells into antibody secreting cells. Depending on the source and type of antigen, T cell independent (TI) and dependent (TD) processes produce plasma cells through distinct cellular pathways where TD antigens are more often associated with long-lived humoral immunity. Although major transcription factor networks and gene expression changes that occur when B cells differentiate to plasma cells have been described, the mechanisms by which these cells adopt a heritable cell fate program and how that program differs between TD and TI immune responses is unknown. Epigenetic processes, which include DNA methylation, control heritable states governing the accessibility of chromatin to transcription factors that mediate differentiation events. Our preliminary data suggest that epigenetic programs are critical for plasma cell fate. In vivo immunization of mice with the TI antigen LPS resulted in a targeted loss of DNA methylation at enhancer regions as splenic B cells differentiated into plasma cells. DNA methylation changes occurred after multiple cell divisions, as did the expression of plasma cell specific genes (Irf4, Blimp1). Conditional B cell deletion of the de novo DNA methyltransferases resulted in a significant increase in TD antigen-specific B cells following immunization, implicating a role for this process in proliferation and/o establishing the plasma cell program. The histone demethylase LSD-1, which decommissions enhancers, was found to be important for this process as well, suggesting that it plays a role in silencing the B cell fate program. Thus, key epigenetic pathways appear to be required for plasma cell fate determination. These pathways and the specific roles for these factors in this process have not been defined. This proposal seeks to fill this void in knowledge. The aims of this study are to: 1) determine the changes in the DNA methylome associated with in vivo plasma cell differentiation to TI and TD antigens; 2) determine the functional dynamics of B cell and plasma cell specific enhancers in plasma cell gene expression programs; and 3) determine the relationship between cell division and plasma cell differentiation in vivo. To accomplish these goals, we have established an efficient set of tools to determine genome wide DNA methylation, chromatin accessibility, histone modifications, and RNA profiles that use small numbers of cells. By integrating these datasets with each other, a robust resource describing the cis-regulatory pathways and matched transcriptional states for TD or TI derived plasma cell will be constructed. This will include bone marrow plasma cells, as these could provide clues into how these cells are epigenetically programmed to provide life-long immunity. Completion of the aims are anticipated to lead to novel and specific targets for improved vaccination and for treatments of disease in which antibody is a component.